During the previous funding period my laboratory developed an in vitro system for the synthesis of enzymatically active reverse transcriptase of duck hepatitis B virus (DHBV), an established model for human hepatitis B virus (HBV). With the help of this system we have already gained substantial new insight into the mechanism by which this polymerase initiates reverse transcription with protein as a primer. These results led tot he proposal of a revised model for hepadnavirus replication, which implicates a sequence on viral RNA, termed epsilon, as the origin of reverse transcription. The scope of our research program now is to exploit our in vitro system for investigations on the biochemical properties and functions of the hepadnavirus DNA polymerase. Specifically, we will investigate the role of host factors, including chaperone hsp90, which appears from preliminary data to be essential for the production of enzymatically active pol gene product. Our observations that host-factors play a critical role for the enzymatic activity of a reverse transcriptase are unprecedented and will provide new insight into first steps of the reverse transcription reaction in hepadnaviruses. Furthermore, we will identify the determinants on the polymerase and on epsilon RNA that control their assembly into a ribonucleoprotein complex, found to be critical for the protein priming reaction and RNA packaging. Since the polymerase does not bear a known RNA recognition motif, results from these investigations will not only contribute to a better understanding of viral replication but also yield new information about determinants that control RNP formation. Additional experiments are proposed for the investigation of the reaction that controls assembly of the polymerase and viral RNA into the nucleocapsid. For this purpose we seek to develop our in vitro polymerase assay into a system that will allow for assembly of intact viral nucleocapsids. Finally, we will investigate the mechanism for the formation of relaxed circular virion DNA into covalently closed circular DNA, which is the first step of the viral DNA replication cycle. Using genetic and biochemical approaches we will determine whether athe viral polymerase or cellular enzymes are required for this reaction and identify the cellular locale in which this reaction occurs. Apart of our overall approach, we will also establish a detailed genetic and functional map of the viral polymerase polypeptide, the availability of which will provide a valuable tool for the successful conduct of the experiments proposed in this application. With the anticipated results obtained through this research program we will not only gain significant information about the mechanism of viral DNA replication, but will further contribute to the identification of novel targets for antiviral therapy. HBV is a pathogen of global significance that can cause acute and chronic hepatitis and induce hepatocellular carcinoma. Whereas primary HBV infection can be prevented by vaccination, so far no treatment is available to over 200 million chronically infected individuals.